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Quest for Quality in Translational Stroke Research—A New Dawn for Neuroprotection?

Despite tremendous progress in modern-day stroke therapy, ischemic stroke remains a disease associated with a high socioeconomic burden in industrialized countries. In light of demographic change, these health care costs are expected to increase even further. The current causal therapeutic treatment paradigms focus on successful thrombolysis or thrombectomy, but only a fraction of patients qualify for these recanalization therapies because of therapeutic time window restrictions or contraindications. Hence, adjuvant therapeutic concepts such as neuroprotection are urgently needed. A bench-to-bedside transfer of neuroprotective approaches under stroke conditions, however, has not been established after more than twenty years of research, albeit a great many data have demonstrated several neuroprotective drugs to be effective in preclinical stroke settings. Prominent examples of substances supported by extensive preclinical evidence but which failed clinical trials are tirilazad and disodium 2,4-sulphophenyl-N-tert-butylnitrone (NXY-059). The NXY-059 trial, for instance, was retrospectively shown to have a seriously weak study design, a trial of insufficient quality and a poor statistical analysis, although it initially met the recommendations of the STAIR committee. In light of currently ongoing novel neuroprotective stroke trials, such as ESCAPE-NA, and to avoid the mistakes made in the past, an improvement in study quality in the field of stroke neuroprotection is urgently needed. In the present review, animal models closely reflecting the “typical” stroke patient, occlusion techniques and the appropriate choice of time windows are discussed. In this context, the STAIR recommendations could provide a useful orientation. Taking all of this into account, a new dawn for neuroprotection might be possible.

Introduction
Stroke is one of the leading causes of death worldwide [1]. The prevalence of stroke was 101 million cases in 2019, and the incident cases were 12.2 million [2]. In the same year, 6.55 million deaths and 143 million disability-adjusted life-years (DALYs) were attributed to stroke [2]. From 1990 to 2019, the total number of prevalent strokes increased by 85%, incident strokes increased by 70%, deaths from stroke increased by 43%, and DALYs due to stroke increased by 32% [2]. In 2019, 62.4% of all strokes were of ischemic etiology [2]. However, in addition to common revascularization treatments, an adjuvant therapy for ischemic stroke is lacking. Hence, several preclinical studies investigating a large number of neuroprotective substances were performed in the last few decades. The neuroprotective strategy, applied alone or in combination, aims at directly inhibiting ischemia-induced cell injury or even cell death [3]. Indeed, numerous substances were shown to have such protective effects in preclinical stroke models at acute, subacute or even delayed time points of the disease. Nevertheless, translational approaches failed until recently. In response, the Stroke Therapy Academic Industry Roundtable (STAIR) published recommendations in order to improve the quality of preclinical stroke studies [4,5]. These criteria included recommendations regarding time windows, reproducibility, animal models, drug dosing, among others. Such recommendations, however, did not result in a paradigm shift in preclinical stroke research, and additional research resources were spent in vain without a successful translational outcome.
In this review, we will first give a brief overview of the pathophysiology of stroke along with preclinical and clinical studies. Thereafter, we will discuss the challenges for bench-to-bedside translation and indicate possible reasons for failure in the past.

Pathophysiology of Stroke
To identify possible neuroprotective agents, a deep understanding of the complex pathophysiology of ischemic stroke is mandatory. In ischemic stroke, impaired perfusion results in an acute glucose and oxygen deficiency, which leads to insufficient anerobic adenosine triphosphate (ATP) production, followed by lactate acidosis and disturbed cellular homeostasis [6]. The latter causes loss of membrane potential with cellular depolarization and the opening of voltage gated Ca 2+ , Na + and K + channels, yielding an inadequate Ca 2+ influx and glutamate-triggered extracellular excitotoxicity [7]. The enhanced intracellular Ca 2+ concentration activates proteases, phospholipases and reactive oxygen species, degrading essential cellular components, such as membrane proteins, cellular lipids and nucleic acids [6]. In parallel, enhanced intracellular Na + influx causes cytotoxic edema.
Apart from excitotoxicity, hypoxia-triggered inflammation is another key factor in stroke pathophysiology [6]. Minutes after hypoxia, neurons release damage-associated molecular patterns which activate resident microglia [8]. Activated microglia, in turn, migrate to the infarct core and to the penumbra and mediate detrimental or protective effects depending on their specific subtype [8]. Furthermore, the secretion of different proinflammatory proteins such as tumor necrosis factor α and interleukins by microglia, neurons and endothelial cells additionally amplifies inflammatory response [8]. The latter enhances the expression of adhesion molecules on endothelial cells and a breakdown of the blood-brain barrier, resulting in extravasation of immune cells into the brain parenchyma [9]. During the first minutes and hours, mainly neutrophils and monocytes enter the brain and exacerbate the inflammatory response [9]. Additionally, T cells can enter the parenchyma and exert detrimental or protective effects [9], thus contributing to neuronal cell loss, mainly through necrosis or apoptosis, although additional and novel forms of cell death are likely to be involved as well.
As a matter of fact, the pathophysiology of ischemic stroke, as briefly outlined, offers a variety of therapeutic targets that range from excitotoxicity to inflammation and various forms of cell death. Elucidating appropriate biologically active agents that may interfere with these signaling cascades constitutes the central strategy for neuroprotective stroke research to this day ( Figure 1).

Preclinical Studies--A Brief Overview
The majority of preclinical stroke studies focus on the acute phase of ischemic stroke. Apart from models of hypoxia with combined oxygen and glucose deprivation (OGD), stroke models with rodents are abundant. The different occlusion techniques to induce cerebral ischemia in these animals are discussed in detail in a later section of this review. Focusing on the acute time window of the disease, a great many studies make use of potentially neuroprotective drugs within six hours after stroke onset. Drugs or biologically active agents that do not cross the blood-brain barrier, e.g., mesenchymal stem cells, are occasionally delivered stereotactically as well [10].
Neuroprotective agents can be classified into major subgroups, among which are free-radical scavengers, glutamate antagonists and anti-inflammatory agents [11]. In addition, stem cells, such as mesenchymal stem cells (MSCs) and stem cell-derived extracellular vesicles, have shown promising preclinical effects [12], although their mode of action appears to focus beyond the subacute time point of stroke. Such preclinical strategies are therefore beyond the scope of the present review, which focuses on (sub-)acute neuroprotection.
The application of free radical scavengers-citicoline or NXY-059, for instance-has been shown to provide substantial neuroprotective effects in preclinical studies but has failed in terms of clinical translation [13][14][15]. Nerinetide is a neuroprotective eicosapeptide which blocks pathological activation of neuronal NO synthetase by inhibition of the NMDAR-postsynaptic density protein (PSD 95) interaction [16]. Its intravenous application resulted in reduced stroke injury, as shown in cultured neurons, rodents and primates [16][17][18]. Similarly, NSP-116, another free radical scavenger, also shows neuroprotective effects under conditions of both preclinical ischemic and hemorrhagic stroke, such that it might be of interest in future clinical trials [19]. In this context, the NMDA receptor antagonist MK801 has been investigated in over 50 preclinical studies where it was found to effectively reduce brain lesion volume and the extent of cerebral edema in stroke rodents [20]. A clinical trial, however, is missing to date. Other glutamate antagonists, such as selfotel, aptiganel and gavestinel, have also showed protective effects in preclinical studies but have proved to be not efficacious in patients with acute ischemic stroke [21,22].
Anti-inflammatory drugs targeting different steps in neuroinflammation, such as microglial activation, interleukin secretion, and immune cell infiltration, have also been

Preclinical Studies-A Brief Overview
The majority of preclinical stroke studies focus on the acute phase of ischemic stroke. Apart from models of hypoxia with combined oxygen and glucose deprivation (OGD), stroke models with rodents are abundant. The different occlusion techniques to induce cerebral ischemia in these animals are discussed in detail in a later section of this review. Focusing on the acute time window of the disease, a great many studies make use of potentially neuroprotective drugs within six hours after stroke onset. Drugs or biologically active agents that do not cross the blood-brain barrier, e.g., mesenchymal stem cells, are occasionally delivered stereotactically as well [10].
Neuroprotective agents can be classified into major subgroups, among which are freeradical scavengers, glutamate antagonists and anti-inflammatory agents [11]. In addition, stem cells, such as mesenchymal stem cells (MSCs) and stem cell-derived extracellular vesicles, have shown promising preclinical effects [12], although their mode of action appears to focus beyond the subacute time point of stroke. Such preclinical strategies are therefore beyond the scope of the present review, which focuses on (sub-)acute neuroprotection.
The application of free radical scavengers-citicoline or NXY-059, for instance-has been shown to provide substantial neuroprotective effects in preclinical studies but has failed in terms of clinical translation [13][14][15]. Nerinetide is a neuroprotective eicosapeptide which blocks pathological activation of neuronal NO synthetase by inhibition of the NMDAR-postsynaptic density protein (PSD 95) interaction [16]. Its intravenous application resulted in reduced stroke injury, as shown in cultured neurons, rodents and primates [16][17][18]. Similarly, NSP-116, another free radical scavenger, also shows neuroprotective effects under conditions of both preclinical ischemic and hemorrhagic stroke, such that it might be of interest in future clinical trials [19]. In this context, the NMDA receptor antagonist MK801 has been investigated in over 50 preclinical studies where it was found to effectively reduce brain lesion volume and the extent of cerebral edema in stroke rodents [20]. A clinical trial, however, is missing to date. Other glutamate antagonists, such as selfotel, aptiganel and gavestinel, have also showed protective effects in preclinical studies but have proved to be not efficacious in patients with acute ischemic stroke [21,22].
Anti-inflammatory drugs targeting different steps in neuroinflammation, such as microglial activation, interleukin secretion, and immune cell infiltration, have also been repeatedly reported. Of note, these substances usually do not exclusively regulate one single inflammatory pathway but have pleiotropic effects instead. Such drugs of interest include tocilizumab, fasudil and veliparib [23][24][25]. Furthermore, many preclinical studies have revealed that transplanted MSCs may modulate post-ischemic immune responses, although such mechanisms imply neurogenesis and angiogenesis rather than genuine neuroprotection [26]. In terms of biological signaling cascades, MSCs appear to mediate neuroprotection or neuroregeneration by partly modulating inflammation through the release of extracellular vesicles (EVs). These small membrane-encapsulated vesicles in the range of nanometers have gained increasing interest in recent years; EVs have been demonstrated to be non-inferior to their host stem cells with regard to their therapeutic potential against stroke [27]. EVs serve as cargo carriers for nucleic acids, proteins and lipids, all of which affect stroke outcomes in their own specific ways [28]. However, EV contents depend on the state of host cell functioning, which determines different metabolic patterns of secreted EVs. As such, microRNA patterns of EVs may change accordingly, and accumulation of specific compounds, such as miR-17-92 or miR-1906, can enhance the therapeutic potential of EVs even further [29,30]. The fact that EVs are able to cross the intact blood-brain barrier after systemic delivery makes them attractive candidates for future stroke therapy [31]. Robust clinical data relating to their therapeutic potential in humans, however, have not yet been generated.

Translational Stroke Studies-A History of Failure
Preclinical evidence of hundreds of substances with neuroprotective features in animal models led to translational clinical trials in some cases. Some examples of these failed bench-to-bedside translations are presented below, together with a short outline of possible reasons for failure.
In over 18 preclinical studies using 544 animals, tirilazad demonstrated promising neuroprotective effects as a free radical scavenger [32]. Tirilazad reduced infarct volumes by 29% and enhanced neurobehavioral scores by 48% on average [32]. This promising evidence resulted in a multicenter, randomized, double-blinded, vehicle-controlled trial of tirilazad mesylate in patients with acute stroke (RANTTAS) [33]. Therefore, 276 patients were treated with tirilazad and 280 patients were given a vehicle within 6 h after stroke onset for 3 days [33]. The study, however, was stopped after the results suggested that tirilazad does not improve neurological outcome as quantified using the Glascow Coma Scale and the Barthel Index [33]. A follow-up study (RANTTAS II) in which higher dosages of tirilazad were used was prematurely stopped by the sponsor when questions regarding safety emerged from a parallel study in Europe (TESS II) [34]. A systematic review investigating the outcome of all clinical trials using tirilazad (four published and two unpublished) revealed that tirilazad treatment actually worsened outcomes in cases of acute ischemic stroke [35]. The systematic review by Sena et al. indicated two major issues regarding this failed translation [32]. First, data from animal studies suggested that tirilazad was effective only in a narrow dose range. Clinical trials with tirilazad, however, used a broad range of doses. If there is only a narrow effective dose range also in humans, this might explain the negative outcomes [32]. Second, in preclinical stroke settings, the interval between stroke onset and treatment was a median of 10 min, whereas in clinical studies it was after 5 h [32]. To make things worse, the preclinical data were not homogeneous, as tirilazad was only effective in lissencephalic but not in gyrencephalic species [32].
Preclinical data for disodium 2,4-sulphophenyl-N-tert-butylnitrone (NXY-059), a free radical spin trap agent, suggested pleiotropic effects on the ischemic cascade leading to neuroprotective effects [36,37]. In contrast to tirilazad, NXY-059 was demonstrated to be neuroprotective in non-human primates, as shown in a trial fulfilling the STAIR criteria [38]. These promising preclinical data, together with evidence of safety, led to two Phase III randomized placebo-controlled trials using NXY-059 within 6 h after stroke onset [39]. In the first trial (SAINT I), a statistical difference was suggested in favor of reduced disability after NXY-059 treatment in patients with acute ischemic stroke [40]. To confirm these observations, a second, much larger, trial (SAINT II), with approximately three thousand patients suffering from acute ischemic stroke, was conducted [40]. However, the SAINT II trial did not confirm the results of the SAINT I trial. Conclusively, NXY-059 was shown to be ineffective for the treatment of acute ischemic stroke within 6 h after the onset of symptoms [40]. Subsequently, AstraZeneca, the company which sponsored the clinical trials, stopped further investigations on NXY-059 in stroke settings.
The failure of the SAINT II trial raised doubts about preclinical evidence generated in animal stroke models and finally about the general concept of neuroprotection as a therapeutic strategy in acute ischemic stroke. A systematic review by MacLeod and colleagues analyzed the preclinical data on NXY-059 in rodent stroke studies [39]. The analysis confirmed that all animal studies, considered together, fulfilled the STAIR criteria. However, there were substantial differences in the quality of individual studies [39]. The authors suggested three factors for the overstated efficacy of NXY-059, namely, publication bias, the potential existence of unpublished negative data and the fact that only one of the preclinical studies fulfilled all the STAIR criteria on its own. In addition, a substantial number of studies had methodical weaknesses, i.e., studies that did not use randomization reported a higher efficacy than studies of higher methodological quality [39]. The authors concluded that the convincing preclinical efficacy was mainly confounded by low study design quality [39]. In line with this, only a minority of studies measured cerebral blood flow during middle cerebral artery occlusion (MCAO) in order to ensure constant infarct volumes [41]. Whereas the SAINT trials used NXY-059 for up to 6 h after stroke onset, the preclinical studies determined a maximum efficacy when NXY-059 is delivered within 4 h [41]. In addition, the stroke population in the SAINT trials displayed heterogeneous stroke patterns, including lacunar and posterior strokes, which did not match the infarct pattern of MCAO animals [41]. Finally, a biodistribution analysis revealed that only small quantities of NXY-059 are able to cross the blood-brain barrier, which raised questions as to the mode of action of NXY-059 [42].
With the failure of the SAINT trials, neuroprotection against stroke appeared to have reached a dead end. Nevertheless, new concepts and strategies have risen from the aforementioned failures of translational stroke trials on neuroprotection. Such findings have led to combined therapeutic approaches, applying both recanalizing and neuroprotective strategies, as represented in the ESCAPE-NA1 trial [43]. More than 1000 patients with ischemic stroke due to large-vessel occlusion were randomized within a 12 h treatment window to receive a single dose of nerinetide (2.6 mg/kg, up to 270 mg) or placebo adjunct to endovascular thrombectomy in this multicenter Phase III trial. Of interest, the authors failed to identify patients with large ischemic penumbra by perfusion-based imaging, despite the hypothesized mechanism of action being that of a "penumbra stabilizer" [44]. The clinical results were disappointing, with similar proportions of the primary endpoint (mRS 0-2 at 90 days) among both groups and no statistical signal of beneficial effects of nerinetide in terms of clinical or radiological secondary outcomes [43]. These negative results of the main analysis seem to be due to the difficulties of translation from-bench-to-bedside. A subanalysis of patients without intravenously administered alteplase (~40%) revealed a surprisingly high benefit of nerinetide treatment, as reflected by improved functional outcomes and by a 7.5% reduction in mortality. Unexpected drug-drug interactions led to reduced plasma-levels of nerinetide in patients with alteplase-treatment and are suspected to be the reason for the lack of treatment effects in this subgroup of patients [43,45]. Further Phase III trials are ongoing to investigate (1) the in-hospital use of nerinetide in ischemic stroke patients undergoing mechanical thrombectomy without prior alteplasetreatment in the 12 h time window (ESCAPE-NEXT; NCT04462536) and (2) the prehospital administration of nerinetide in patients with suspected stroke (FRONTIER; NCT02315443). A preplanned analysis of non-thrombolyzed ischemic stroke patients of ESCAPE-NA1 and ESCAPE-NEXT will clarify whether nerinetide represents the first clinically relevant neuroprotectant for ischemic stroke patients.

Challenges and Perspectives for Bench-to-Bedside Translation
Given the fact that bench-to-bedside translation has failed to this day, possible sources of errors must be identified and eliminated. Consequently, for successful bench-to-bedside translation, aspects such as (1) the general STAIR recommendations, (2) animal models, (3) occlusion techniques, (4) as well as time windows and possible side effects, should be considered.

STAIR Recommendations
The STAIR recommendations, initially published in 1999, were intended to improve the quality of preclinical studies investigating possible novel stroke therapies [5]. According to the initial STAIR recommendations, adequate dose-response curves, defined time windows in well-characterized models, blinded and controlled reproducible studies, assessment of histological and functional outcomes, and permanent vessel occlusion followed by transient occlusion should be provided (overview in Figure 2). In addition, initial rodent studies should be followed by studies in gyrencephalic species. The aspects of dose-response curves, therapeutic time windows, outcome measures, multiple species use and reproducibility were revised in an updated version but remained valid [4]. To fulfil reproducibility, experiments should be replicated in at least one or more independent laboratory. The fundamentals of good scientific inquiry should also be applied by implementing randomization and eliminating outcome assessment bias, reporting the reasons for excluding animals from final data analyses and defining inclusion/exclusion criteria, performing power analysis and sample size calculations, and declaring any relevant conflicts of interest [4]. However, these criteria are not binding, and a vast number of studies still do not consider them fully.

Challenges and Perspectives for Bench-to-Bedside Translation
Given the fact that bench-to-bedside translation has failed to this day, possible sources of errors must be identified and eliminated. Consequently, for successful benchto-bedside translation, aspects such as (1) the general STAIR recommendations, (2) animal models, (3) occlusion techniques, (4) as well as time windows and possible side effects, should be considered.

STAIR Recommendations
The STAIR recommendations, initially published in 1999, were intended to improve the quality of preclinical studies investigating possible novel stroke therapies [5]. According to the initial STAIR recommendations, adequate dose-response curves, defined time windows in well-characterized models, blinded and controlled reproducible studies, assessment of histological and functional outcomes, and permanent vessel occlusion followed by transient occlusion should be provided (overview in Figure 2). In addition, initial rodent studies should be followed by studies in gyrencephalic species. The aspects of dose-response curves, therapeutic time windows, outcome measures, multiple species use and reproducibility were revised in an updated version but remained valid [4]. To fulfil reproducibility, experiments should be replicated in at least one or more independent laboratory. The fundamentals of good scientific inquiry should also be applied by implementing randomization and eliminating outcome assessment bias, reporting the reasons for excluding animals from final data analyses and defining inclusion/exclusion criteria, performing power analysis and sample size calculations, and declaring any relevant conflicts of interest [4]. However, these criteria are not binding, and a vast number of studies still do not consider them fully.

Animal Models
Essential for a successful bench-to-bedside translation is the use of appropriate animal models. For stroke models, rats and mice are by far the most commonly used animals, although rabbits and non-human primates are becoming increasingly popular [46]. Most of the rats or mice used in stroke models are young, male animals without any typical comorbidities, such as hypercholesterinemia, arterial hypertension, diabetes or obesity. Thus, these animals are more stress-resistant and healthier in contrast to most stroke patients. This mismatch between stroke patients and animal models may affect individual outcomes significantly. However, different animal models have been introduced to better mimic stroke patients. For example, hypercholesterinemia is reflected by using apolipoprotein-E (ApoE)−/−, LDL receptor−/− or human ApoB transgenic rodents [47]. Plasma cholesterol levels can be further increased by placing rodents on a western diet [48].

Animal Models
Essential for a successful bench-to-bedside translation is the use of appropriate animal models. For stroke models, rats and mice are by far the most commonly used animals, although rabbits and non-human primates are becoming increasingly popular [46]. Most of the rats or mice used in stroke models are young, male animals without any typical comorbidities, such as hypercholesterinemia, arterial hypertension, diabetes or obesity. Thus, these animals are more stress-resistant and healthier in contrast to most stroke patients. This mismatch between stroke patients and animal models may affect individual outcomes significantly. However, different animal models have been introduced to better mimic stroke patients. For example, hypercholesterinemia is reflected by using apolipoprotein-E (ApoE)−/−, LDL receptor−/− or human ApoB transgenic rodents [47]. Plasma cholesterol levels can be further increased by placing rodents on a western diet [48]. Indeed, brain injury after ischemic stroke has been shown to be exacerbated in ApoE−/− mice, demonstrating the relevance of using such models [49]. Mimicking hypertension, the most prevalent risk factor for stroke [50], can be achieved by using primary hypertensive or salt-sensitive rats that display heightened immune responses poststroke, which leads to enlarged infarct volumes [51,52]. Likewise, some models induce hypertension by clipping one or both kidney arteries, resulting in systolic blood pressure values of 200-225 mmHg [53].
Diabetes increases the risk of stroke up to 3 times [54]. Models with spontaneously diabetic animals or induced diabetes are used in preclinical studies [55]. King gave an extensive overview of the different diabetes animal models in his review [56]. For example, the repeated application of Langerhans β cell toxin, which leads to a loss of β cells in the pancreas, is a widely used technique for inducing type 1 diabetes in animal models [56]. Type 2 diabetes can be triggered with a similar approach when β cell toxin is applied just once [56]. Obesity and diabetes often go hand in hand in clinical settings, and therefore animals that are deficient for leptin and the leptin receptor are an elegant way of modeling such a setting [56]. Leptin deficiency leads to hyperphagia in mice, resulting in obesity and hyperglycemia at the age of 4-8 weeks [56]. Not surprisingly, in obese and diabetic mice poststroke, long-term motor recovery is significantly impaired [57]. Future studies must therefore focus more on comorbidity models to reflect the clinical situation more adequately.

Occlusion Techniques
Ischemic stroke is characterized by the occlusion of brain-supplying arteries, resulting in decreased cerebral blood flow. Various stroke model techniques are used to mimic this insult in animals. Selecting an appropriate model is crucial for the significance of the results in relation to future translations. The following five techniques are most used: (1) intraluminal occlusion, (2) transcranial occlusion, (3) cerebral photothrombosis, (4) endothelin-1 occlusion and (5) cerebral embolism. Each technique has advantages and disadvantages which will be discussed in the following paragraphs (summarized in Table 1). The most widely used experimental stroke technique is the intraluminal MCAO model [46]. In this model, a monofilament is inserted into the common carotid and then pushed forward to the internal carotid until the MCA branches off [58]. Thus, cerebral blood flow is decreased to 10-20% but rapidly restored after removal [58]. The occlusion time determines the extent of ischemic damage, which varies between 10 and 120 min [58]. This results in a large infarct core surrounded by a well-defined penumbra [59]. On account of the latter, the model is suitable for neuroprotective studies. However, to create reproduceable insults, in vivo measurement of cerebral blood flow during the occlusion is essential [59]. The lack of cerebral blood flow measurement may be one factor for false results in the past, together with the fact that such a model does not include the presence of a thrombus. Studies on thrombolysis are therefore not feasible in this model.
Using transcranial occlusion, the MCA can be occluded permanently or transiently across a cranial window [60]. Techniques such as coagulation, clip or suture find use and are often combined with ipsilateral common carotid artery occlusion [59]. On the one hand, the latter allows the generation of large brain infarcts with pronounced neurological deficits [59]. On the other hand, it is possible to produce smaller infarcts in comparison to intraluminal occlusion along with a well-defined penumbra. A potential weakness is that variants of cerebrovascular anatomy are not correctly identified, and the exact placement of MCA occlusion is insufficiently standardized [59].
The model of photothrombosis includes the systemic delivery of a photosensitive dye that is transcranial-illuminated, resulting in thrombus formation that locally occludes microvessels [61]. The advantage of this technique is the possibility of producing precise and well-defined infarcts in specific regions by stereotactic precision [61]. This model has disadvantages, too, however, since lesions are located superficially, with no genuine penumbra, and hardly react to thrombolytic drugs.
Endothelin-1 is a long-acting vasoconstrictive peptide which is administered directly to an exposed vessel, via stereotactical injection, or delivered on the cortical surface only [62]. The endothelin-1 injection results in a significant drop in cerebral blood flow that lasts over a period of hours followed by reperfusion [63]. The advantage of this model lies in its having extremely low mortality rates as a consequence of being a less invasive technique. However, since endothelin-1 has been shown to induce astrocytosis and to facilitate axonal sprouting on its own, this could lead to the misinterpretation of experiments evaluating poststroke neural repair [64].
Cerebral embolism is the most common etiology of ischemic stroke and is therefore, in respect to future clinical translation, highly relevant. There are mainly three different types of cerebral embolism models used in rodents, i.e., synthetic macrospheres or microspheres, autologous blood clots and stereotactic thrombin delivery [59]. All of these models create a well-defined penumbra and are on this account well-suited to study neuroprotective drugs administered alone as well as in combination with thrombolytic agents [62]. However, these models produce highly variable infarcts compared to intraluminal MCAO models and are associated with poor long-term animal survival [59].

Therapeutic Time Window
In a substantial number of preclinical neuroprotective studies, the therapeutic agents were given prior or immediately after the induction of cerebral ischemia. This does not reflect the clinical situation. As such, it is mandatory to generate evidence for drugs given within a reasonable time window if bench-to-bedside translation is desired. Neuroprotective drugs play a key role in translation, but in preclinical studies they have generally been found to have a short therapeutic time window, impairing clinical translation even further [11]. NXY-059, for example, discussed in detail above, has been shown to have a maximum therapeutic window of 4 h in preclinical studies. In clinical trials, however, patients received NXY-059 up to 6 h poststroke, which could be one explanation for the failure of the clinical trial [41].

Conclusions and Perspective
Stroke is one of the leading causes of death and disability worldwide. Hence, there is an urgent need to develop neuroprotective strategies. Ischemic stroke is characterized by a complex pathophysiology involving excitotoxicity, inflammation and various forms of cell death. Studying appropriate biologically active agents that may interfere with these signaling cascades, especially in the acute phase of stroke, constitutes the central strategy of neuroprotective stroke research, past and present. Such agents can be classified into major subgroups, among which are free-radical scavengers, glutamate antagonists and anti-inflammatory agents. In addition, stem cells such as MSCs and stem cell-derived extracellular vesicles have shown promising preclinical effects. However, despite enormous research efforts in the past, no effective therapy has been discovered.
Possible reasons for the continuing failure are statistical weaknesses in studies, nontransferable study designs and overall low study quality. Conclusively, an improvement in the quality of preclinical neuroprotection studies is needed. This requires the use of animal models reflecting the typical stroke patient, occlusion methods that properly match research questions and, finally, therapeutic times windows that are transferable to the clinical setting. Hence, the STAIR recommendations may give a useful orientation. Taking this into account, a new dawn for neuroprotection may be possible.

Conflicts of Interest:
The authors declare no conflict of interest.

CBF
cerebral blood flow ICA internal carotid artery CCA common carotid artery MCA middle cerebral artery


Dominant Components of the Giant Panda Seminal Plasma Metabolome, Characterized by 1H-NMR Spectroscopy

Simple Summary As China’s flagship animal, the giant panda (Ailuropoda melanoleuca) attracts much attention due to its small population and low natural reproductive rate. Therefore, artificial insemination has become the leading practical approach in the captive breeding programs of giant pandas worldwide. Seminal plasma acts as a medium between spermatozoa and the external stimuli, and its characteristics have been directly linked to fertility in both artificial insemination and natural fertilization. The current work, for the first time, attempts to characterize, by proton magnetic resonance spectroscopy (1H-NMR), the metabolome of healthy giant panda seminal plasma. A total of 35 molecules were quantified, with distinct age-related trends highlighted by a multivariate analysis, and the concentrations of 2,3-butanediol were significantly different between individuals younger than 8 years and older than 13 years. In addition, isopropanol’s concentration was significantly linked to estrus stages. Besides, the variations in the metabolome’s profile with storage time were also evaluated. This study may serve as a reference for research wishing to shed light on the biological mechanisms affecting giant panda sperm’s overall quality and may ultimately lead to novel approaches to giant panda artificial insemination. Abstract As an assisted breeding technique, artificial insemination has become the main effective practical approach in the captive breeding programs of giant panda worldwide. The composition of seminal plasma plays an important role in the success of breeding. The present work is the first attempt to characterize, by proton magnetic resonance spectroscopy (1H-NMR), the metabolome of healthy giant panda seminal plasma. A total of 35 molecules were quantified, with the concentration of 2,3-butanediol being significantly different between individuals younger than 8 years and older than 13 years, and other distinct age-related trends were highlighted by a multivariate analysis. Isopropanol’s concentration was significantly linked to estrus stages. Besides, the variations in the metabolome’s profile during storage were also evaluated. This study may serve as a reference for further research wishing to shed light on the biological mechanisms affecting giant panda sperm’s overall quality and may ultimately lead to novel approaches to giant panda artificial insemination.

Introduction
As the flagship animal of China, the giant panda (Ailuropoda melanoleuca) is one of the most endangered mammals on earth, which is attributed to its small population and limited reproductive rate [1]. Female giant pandas are in estrus for only a few days each year, so less than 10 percent of giant panda males mate, and less than 30 percent of females conceive naturally [2,3]. Therefore, artificial insemination has become the main practical approach in the captive breeding programs of giant pandas worldwide since it was first attempted in the 1970s [4][5][6].
To maximize the success rate of the procedure, the spermatozoa number, motility, and morphology have been widely evaluated [2,7,8]. As seminal plasma acts as a medium between spermatozoa and the external stimuli, its characteristics have also been directly linked to fertility [9]. Recent advances in high-throughput techniques (GC-MS (gas chromatography-mass spectrometry), LC-MS (liquid chromatograph-mass spectrometry), and 1 H-NMR (proton magnetic resonance spectroscopy)) have shed light on the role played by its metabolome, the ensemble of its low-weight molecules. Metabolomics, the study of the metabolome, has therefore become a valuable tool for studying humans' and other mammals' seminal plasma [10][11][12]. For instance, Jayaraman et al. successfully distinguished different forms of male infertility through an NMR-based metabolomic approach and identified lysine as a potential biomarker for the detection and diagnosis of idiopathic infertility [12]. Mumcu et al. found potential biomarkers of infertility by studying the metabolome of seminal plasma in patients with idiopathic oligoasthenoteratozoospermia [13].
In humans and in any other animal, the age of the donor has been reported to be a key factor for seminal plasma perturbations, so the relationships between seminal plasma characteristics and age have been widely investigated. For instance, Fosse et al. found that older men exhibit a high pro-inflammatory seminal plasma profile and determine unexplained recurrent pregnancy losses [14]. Vince et al., studying bulls during cold and warm periods of the year, found age-related differences in the antioxidant profiles of their seminal plasma [15]. Such findings agree with those of Sicy et al., who assessed the seminal plasma oxidant/antioxidant status of Arab stallions of different ages [16]. In rams, Souza et al. found that the total protein concentration in the seminal plasma significantly increased during the first 28 weeks of life [17]. In Arabian horses, Ahmed et al. noticed significant age-related effects on seminal plasma's biochemical constituents, namely, glycerylphosphorylcholine, ergothioneine, and total protein concentrations [18].
Interestingly, the estrus stages of the female giant panda have been found to affect male giant panda semen quality, both directly and indirectly. Tsutsui et al. found that male giant panda semen volume, sperm count, and testis volume markedly differed from 90 days before estrus until 66 days after estrus when a female giant panda was housed nearby. From this observation, they inferred that spermatogenesis was active in this male panda from approximately 3 months before estrus to 2 months after estrus in the adjacent female [3]. In addition, Palmer et al. reported that testes morphometry, fecal androgen excretion, seminal quality, and the panda's overall behavior clearly demonstrated reproductive seasonality in the male giant panda [19].
Cryopreservation is considered to be the main form of sperm preservation and is widely used for artificial insemination. It is known that plasma characteristics may be altered during storage with consequences on spermatozoa motility [20]. However, most of the works on the topic have focused on the effects of dilution or removal, the most common practice prior to cryopreservation in most mammalian species [21]. Lower attention has been paid to the detailed study of the effects of storage time on the metabolome of seminal plasma.
The above works suggest that advances in the procedures for the artificial insemination of giant pandas may come from the study of the seminal plasma metabolome, which is the object of the present work. With the purpose of providing reliable quantitative information, an untargeted approach based on 1 H-NMR was used by taking advantage of the high reproducibility of this analytical platform [22]. As the samples we were able to collect had  been frozen and donated from animals covering all life stages of the giant panda, we had  the opportunity to obtain preliminary information on how age, the estrus stages of the  female giant panda, and storage time could influence the molecular profile of male giant  panda seminal plasma. These findings may serve as a starting point for further research shedding light on the biological mechanisms impacting giant panda sperm's overall quality and ultimately lead to novel approaches to giant panda artificial insemination.

Sample Collection
All samples were kindly provided, regardless of the purpose of this study, during the procedures for artificial insemination and cryogenic storage that are routinely conducted at the China Conservation and Research Center for the Giant Panda by selecting for metabolomics investigation an aliquot of sperm stored for insemination. Sperm freezing medium (TEST-yolk buffer with glycerol and gentamicin) and sperm refrigeration medium (TEST-yolk buffer with gentamicin) were added immediately after sample collection. Semen collection was achieved with the panda in dorsal recumbency. Prior to electroejaculation, feces were manually removed from the rectum, and the penis and surrounding area were washed with warm water. A 4.2 cm diameter, 20 cm long probe was used with three l cm circular electrodes. The probe was placed in the rectum to a depth of 15-20 cm. Electrostimulations were performed with a 110 V, 60 Hz machine with a 220 V, 50 Hz transformer. A range of 5-12 stimulations with 2-8 V (a maximum of 200 mA) over a 3-5 s period was repeated to achieve ejaculation. Semen was collected into a plastic container and immediately frozen with liquid nitrogen. All the samples were shipped to a laboratory in less than 2 h with ice immediately after collection. Samples collections were performed from 2010 to 2019 and stored at −80 • C until analysis.
Immediately before 1 H-NMR analysis, seminal plasma samples were thawed and centrifuged for 15 min at 18,630 g at 4 • C. Finally, 300 µL of supernatant was mixed with 300 µL of bi-distilled water and 100 µL of NMR analysis solution for 1 H-NMR analysis.
The 1 H-NMR spectra were obtained by means of an AVANCE III spectrometer (Bruker, Chengdu, China) operating at a frequency of 800.30 MHz at 298 K and equipped with the software Topspin 3.5. Following Zhu et al. [24], the signals from broad resonances originating from macromolecules were suppressed by a CPMG filter composed of 400 echoes with a τ of 400 µs and a 180 • pulse of 30.48 µs, for a total filter of 332 ms. The HOD residual signal was suppressed by means of presaturation. This was performed by employing the cpmgpr1d sequence, part of the standard pulse sequence library. Each spectrum was acquired by summing 64 transients using 64 K data points over a 16,025.64 Hz spectral window with an acquisition time of 2.025 s.
The spectra phase was manually adjusted in Topspin, while the subsequent adjustments were performed in R computational language by means of a script that was developed in-house [25]. After the residual water signal removal, the 1 H-NMR spectra were baseline-corrected through peak detection, in accordance with the "rolling ball" principle [26] implemented in the R package named baseline [27]. The chemical shift and multiplicity of signals allowed the molecules' annotation by taking advantage of the Chenomx software library (ver. 8.3, Chenomx Inc., Edmonton, AB, Canada).
In order to remove the potential confounding factor of dilution [28], the ratio between the area of TSP signal and the total spectrum intensity of each sample was calculated. This allowed us to estimate the median unspecific change in metabolites caused by water and to Animals 2022, 12, 1536 4 of 14 select the sample characterized by the mostly representative dilution. The molecules were quantified in this sample by employing the added TSP as an internal standard, while any other sample was then normalized by means of probabilistic quotient normalization [28], which was set up to exclude spectra portions ascribed to water and glycerol. The integration of the signals was performed for each molecule by means of rectangular integration.

Statistical Analysis
The statistical analysis was conducted in R computational language and online data analysis platform MetaboAnalyst 5.0 (https://www.metaboanalyst.ca, accessed on 1 March 2022). Prior to the univariate analyses, the distribution of the concentrations of the molecules was brought to normality according to Box and Cox [29]. To demonstrate perturbations caused to single molecules by the effects considered, t-tests were applied with a significance limit of p < 0.05. To demonstrate perturbations encompassing the metabolome's profile in its entirety, robust principal component analysis (rPCA) models [30] and heatmaps were employed. The outcome of each rPCA model was constituted by a scoreplot, the projection of the samples in the PC space. This allowed us to highlight the underlying structure of the data. Besides, we calculated Pearson's correlation plot, relating the concentration of each variable to its importance over each component of the model.

Characterization of Samples
Samples from 15 healthy captive giant pandas were obtained (Table 1). According to Zhu et al. and Wei et al. [31,32], all the giant pandas were considered to be divided into three groups according to mating age. On average, wild giant pandas come to estrus at 7.5 years and mating age starts at 8.5 years, while captive giant pandas come to estrus earlier than wild ones. In addition, captive giant pandas are considered to be adults when their age is between 5.5 and 20. Therefore, adult phase I comprised animals younger than 8 years of age, adult phase II comprised those between 8 and 12 years of age, and adult phase III comprised animals older than 13. The estrus of the giant panda begins from March to May, so the samples collected in February and March were considered pre-estrus (PRE), those collected in April were considered middle-estrus (MID), and those collected in May and June were considered post-estrus (POS). Focusing on cryogenic storage time, all the samples were considered to pertain to the long storage group when stored for more than 6 years, while they were considered as pertaining to the short storage group otherwise.

Characterization of Semen Plasma Metabolome of Giant Panda
In giant panda seminal plasma samples, we were able to characterize 35 metabolites. The details of the characterization by 1 H-NMR are visually represented in Figure 1 through a typical 1 H-NMR spectrum of the collected samples. The complete list of molecules and their concentrations is reported in the Table 2.   G11  13  February 2012  G12  18  April 2010  G13  13  April 2018  G14  14  June 2018  G15  14 June 2018

Characterization of Semen Plasma Metabolome of Giant Panda
In giant panda seminal plasma samples, we were able to characterize 35 metabolites. The details of the characterization by 1 H-NMR are visually represented in Figure 1 through a typical 1 H-NMR spectrum of the collected samples. The complete list of molecules and their concentrations is reported in the Table 2. To ease the reader's visual inspection, for each portion, a spectrum with a convenient signal-to-noise ratio has been selected.  The characterized molecules mainly pertained to the classes of carbohydrates and derivatives, organic acids and derivatives, amino acids, peptides and derivatives, and nucleosides, nucleotides, and analogues. The relative abundances of those classes in the samples from the three panda's groups can be visually inspected in Figure 2. The overall relative concentrations of amino acids, peptides, and derivatives showed increasing trends with age, specifically, 24.76%, 27.22%, and 29.06% in three groups, respectively. On the contrary, the contents of organic acids and derivatives were negatively related to age, namely, 38.93%, 38.75%, and 36.22%, respectively. Among the quantified molecules, the concentrations of sn-glycerol-3-phosphocholine, lactate, citrate, and carnitine were the most represented in each group. The characterized molecules mainly pertained to the classes of carbohydrates and derivatives, organic acids and derivatives, amino acids, peptides and derivatives, and nucleosides, nucleotides, and analogues. The relative abundances of those classes in the samples from the three panda's groups can be visually inspected in Figure 2. The overall relative concentrations of amino acids, peptides, and derivatives showed increasing trends with age, specifically, 24.76%, 27.22%, and 29.06% in three groups, respectively. On the contrary, the contents of organic acids and derivatives were negatively related to age, namely, 38.93%, 38.75%, and 36.22%, respectively. Among the quantified molecules, the concentrations of sn-glycerol-3-phosphocholine, lactate, citrate, and carnitine were the most represented in each group.

Metabolome's Age-Related Features
To evaluate the overall molecular profile trends with age, a robust principal component analysis (rPCA) model ( Figure 3) and a heatmap (Figure 4) were set up.

Metabolome's Age-Related Features
To evaluate the overall molecular profile trends with age, a robust principal component analysis (rPCA) model ( Figure 3) and a heatmap ( Figure 4) were set up.
In terms of the rPCA model, the first component represented 76.2% of the variance, summarizing the peculiarities connected to age, with individuals pertaining to adult phase I and III appearing at the low and high PC scores, respectively, and adult phase II individuals at intermediate values. The loading plot of Figure 3B shows that samples from adult phase III were characterized by higher concentrations of ethanolamine, TMAO, 2,3-butanediol, aspartate, and creatinine, while samples from adult phase I showed higher concentrations of ethanol, tyrosine, glucose, citrate, betaine, and phenylalanine ( Figure 3B). The common trends of ethanol, tyrosine, glucose, citrate, betaine, and phenylalanine can be best appreciated through a heatmap, as shown in Figure 4. The same applies to the common and opposite trends of ethanolamine, TMAO, 2,3-butanediol, aspartate, and creatinine.     A comparison between adult phase I and III individuals by a t-test highlighted a significant difference in 2,3-butanediol, as shown in the volcano plot in Figure 5.
phase I and III appearing at the low and high PC scores, respectively, and adult phase II individuals at intermediate values. The loading plot of Figure 3B shows that samples from adult phase III were characterized by higher concentrations of ethanolamine, TMAO, 2,3butanediol, aspartate, and creatinine, while samples from adult phase I showed higher concentrations of ethanol, tyrosine, glucose, citrate, betaine, and phenylalanine ( Figure  3B). The common trends of ethanol, tyrosine, glucose, citrate, betaine, and phenylalanine can be best appreciated through a heatmap, as shown in Figure 4. The same applies to the common and opposite trends of ethanolamine, TMAO, 2,3-butanediol, aspartate, and creatinine.
A comparison between adult phase I and III individuals by a t-test highlighted a significant difference in 2,3-butanediol, as shown in the volcano plot in Figure 5. In addition, we calculated Pearson's correlation between the age and the concentration of each molecule we quantified. The result showed that a significant positive correlation existed only for 2,3-butanediol (r = 0.61 and p = 0.02).

Giant Panda Seminal Plasma Metabolome Is Related to the Estrus Phases
In parallel with age, a univariate analysis ( Figure 6) was set up to study the overall metabolome's trends connected to estrus. A heatmap was also set up (Figure 7), to observe the molecules' trend similarities. In addition, we calculated Pearson's correlation between the age and the concentration of each molecule we quantified. The result showed that a significant positive correlation existed only for 2,3-butanediol (r = 0.61 and p = 0.02).

Giant Panda Seminal Plasma Metabolome Is Related to the Estrus Phases
In parallel with age, a univariate analysis ( Figure 6) was set up to study the overall metabolome's trends connected to estrus. A heatmap was also set up (Figure 7), to observe the molecules' trend similarities.    As shown in Figure 6, one molecule, namely isopropanol, exhibited significantly higher levels post-estrus than pre-estrus. The heatmap allows us to appreciate that a list of molecules exhibited higher concentrations in the pre-estrus group, such as formate and galactarate. Other molecules, such as isopropanol and 1,3-dihydroxyacetone, were more concentrated in the post-estrus group.

Discussion
Seminal plasma is a complex biological fluid that is mainly characterized by inorganic ions, low-molecular-weight organic compounds, peptides, and hormones [33]. This organic and inorganic complex mixture is secreted from different endocrine glands and is the functional reflective index of the corresponding glands [34]. In addition, the metabolites present in seminal plasma play several roles related to sperm function, such as energy production, motility, protection, pH control, and the regulation of metabolic activity [35]. To the best of our knowledge, this is the first attempt to acquire metabolomic data from giant panda seminal plasma. A total of 35 molecules were unequivocally quantified, a number much higher than previously obtained in similar studies on other animals [36] or humans [37]. With the kind help of the China Conservation and Research Center for the Giant Panda, we had the opportunity to investigate the effects of age and estrus on giant panda seminal plasma. Age has been widely regarded to be a key factor for semen or/and seminal plasma perturbations. For instance, Chen et al. found, in humans, that semen volume, spermatozoa concentration, total spermatozoa count, motility, total motile sperm, and morphology significantly decreased with age [38]. In the current study, an rPCA model based on the entire set of molecules quantified highlighted distinct trends connected to age. A comparison between age adult phase I and III and Pearson's correlation showed a specific effect of age on the concentration of 2,3-butanediol. The presence of this molecule in mammalian seminal fluid was recently confirmed by some of the authors of the present work [39] while studying horse's seminal plasma. It is interesting to notice that 2,3-butanediol is a catabolite of pyruvate, which could not be quantified in the present work due to the superimposition of its signal with some from glutamate. [40] Pyruvate is considered to be one of the major sources of energy for spermatozoa [41]. Higher concentrations of 2,3-butanediol in older individuals could therefore indicate a lower concentration of pyruvate, with detrimental effects on sperm quality. On the other hand, in light of the cryogenic storage practice of semen, it is worth noticing the potential protective role of this molecule, as shown by Jeyendran et al. on bovine spermatozoa [39].
Among the molecules showing trends connected to an animal's age or estrus phase, aspartate has been demonstrated, with particular reference to its D enantiomer, to have an impact on a number of key parameters determining sperm quality [42][43][44]. 1 H-NMR with water as a solvent cannot separately quantify the D and L enantiomers. However, Macchia et al. noticed that administering rabbits with increasing doses of DL-aspartate caused an increase in the D-aspartate concentration, while the concentration of L-aspartate did not vary [45]. It is therefore tempting to speculate that the higher concentration of aspartate we found in younger individuals witnessed a higher concentration of its D enantiomer with its consequent positive effects, which include increased sperm motility and concentration.
One of the main sugars in the seminal plasma is glucose, which is essential for ATP production and the motility of the spermatozoa [46]. No significant correlation has been found between glucose concentration and fertility in bulls [47]. This finding was confirmed by Cevik et al., even though a significantly higher level of glucose was found in normozoospermic bulls than in oligoasthenozoospermic bulls [48]. Gupta et al. revealed that the concentration of phenylalanine in the seminal plasma of humans can be used as a biomarker to determine infertility [33]. Engel et al. found that the concentrations of phenylalanine in seminal plasma were positively correlated with the volume of the ejaculate, reaching statistical significance. In addition, high creatinine levels were significantly positively correlated with the amount of immotile sperm [49]. This finding agrees with an NMR-based study that showed increased creatinine levels in the seminal plasma of patients with asthenozoospermia [37]. Phenylalanine is an essential aromatic amino acid. Once in the body, most of the phenylalanine is usually converted to tyrosine, which in turn is degraded to levodopa and then to dopamine [50]. Creatinine, a degradation product of creatine, is one of the most abundant biogenic amines in blood serum [23] and is cleared via the kidneys [51]. In the present context, it is worth mentioning that there are reports of elevated creatine kinase activity in subfertile patients [52,53]. Citrate is the main anion of seminal plasma, where it chelates calcium ions and limits sperm capacitation and spontaneous acrosome reactions [54]. Citrate and acid phosphatase can be considered to be markers for prostate activity [55]. Kumar et al. found that high-fertility bulls exhibited low levels of citrate [56]. Such findings are consistent with similar studies in infertile men [57].
The effects of storage time on the metabolome of biomaterials (i.e., urine and feces) have been evaluated in a limited number of papers [58,59], none of which focused on potential changes in giant panda seminal plasma with time. At present, both fresh and thawed frozen giant panda semen samples are successfully employed for AI. A real-life example is represented by the giant panda Mei Xiang, who was inseminated with frozen semen collected 14 years before and recently gave birth to Bao Bao (https://nationalzoo.si. edu/animals/news/how-artificial-insemination-helps-boost-panda populations, accessed on 15 March 2022). Our preliminary data indicate that there are indeed metabolomic changes connected to storage. Whether these modifications would affect the success rate of AI still needs further investigations.
The rarity of giant pandas has limited the availability of samples for the present investigation. To adapt to this limitation, among the various multivariate methods described to highlight the main trends underlying the data, we selected rPCA, an unsupervised method that is not prone to overfitting. This method allowed us to exclude the presence of potential outliers.

Conclusions
To the best of our knowledge, this is the first attempt to acquire metabolomic data of giant panda seminal plasma by means of 1 H-NMR. We were able to characterize 35 molecules in giant panda seminal plasma and to illustrate, with the limits represented by a low number of samples, the effects of age, estrus stages, and storage time on the seminal plasma metabolome, with particular reference to 2,3-butandiol, isopropanol, galactarate, and ethanolamine. For some of them, such as 2,3-butandiol and aspartate, we were able to suggest direct links with sperm quality, thus offering possibilities for novel approaches to giant panda artificial insemination. Institutional Review Board Statement: Ethical review and approval were waived for this study as the samples were collected regardless of the purpose of this study during the procedures for artificial insemination and cryogenic storage that are routinely conducted at the China Conservation and Research Center for the Giant Panda.


Inhibitory effects of saeu‐jeot extract on NLRP3 inflammasome activation and radiation‐induced micronucleus formation

Abstract Purpose Saeu‐jeot, or shrimp paste, is a traditional Korean high‐salt fermented seafood. This study elucidated the effects of saeu‐jeot extract (SJE) on NLRP3 inflammasome activation and radiation‐induced micronucleus formation. Method We treated lipopolysaccharide‐primed mouse bone marrow‐derived macrophages with the NLRP3 inflammasome activators ATP and nigericin. We also analyzed the acute effects of ionizing radiation on micronucleated polychromatic erythrocytes (MnPCEs) in whole‐body gamma‐irradiated male BALB/c and C57BL/6 mice after oral administration of SJE. Results SJE significantly inhibited NLRP3 inflammasome activation, reducing IL‐1β secretion in vitro. In addition, the frequency of MnPCEs was significantly lower in SJE‐treated mice. Conclusions SJE has anti‐inflammatory effects and reduces radiation‐induced chromosome damage. Advance in knowledge: There are no reports concerning the effects of SJE on NLRP3 inflammasome activation or radioprotection. This experiment showed the radioprotective effects of saeu‐jeot.

Gamma radiation can increase ROS levels, resulting in IL-1β expression. ROS are presumed to activate NLRP3 by promoting the dissociation of thioredoxin-interacting protein (TXNIP) from thioredoxin (TRX), which allows TXNIP to bind directly to and activate NLRP3 (Haneklaus et al., 2013;Licandro et al., 2013). Inflammation has a pivotal role in modulating radiation responsiveness; inflammatory mediators act together to perpetuate and amplify the inflammatory cascade. These mediators suppress DNA repair, leading to microsatellite and chromosome instability, which culminate in abnormal chromosome segregation and aneuploidy (Multhoff & Radons, 2012). Micronucleus formation is a sign of this abnormal chromosome segregation. We previously studied the inhibition of radiation-induced micronucleus formation by Korean traditional fermented soybean food (Kim et al., 2019). Saeu-jeot, a traditional Korean high-salt fermented seafood, consists of tiny shrimp that have been fermented for several months with sea salt. Recently, the therapeutic effects of SJE have been evaluated (Koo et al., 2016); however, there are no reports concerning the effects of SJE on NLRP3 inflammasome activation or radioprotection. Therefore, this study assessed the effects of SJE on NLRP3 inflammasome activation in vitro and on radiation-induced micronucleus formation in vivo.

| Differentiation of bone marrow-derived macrophages (BMDMs)
Primary BMDMs were cultured and differentiated in vitro; treated lipopolysaccharide-primed mouse BMDMs with the NLRP3 inflammasome activators ATP and nigericin (NG). Briefly, BMDMs were derived from femoral and tibial bone marrow progenitors.

| Western blot analysis
Supernatants, lysates, and pellet samples were separated by SDS-PAGE using 10% or 16% polyacrylamide gels. After electrophoresis, the separated proteins were transferred to PVDF membranes (Pall Co., NY, USA). The membranes were probed overnight at 4°C with anti-mouse IL-1β antibody (R&D Systems, MN, USA) or anti-actin antibody (Santa Cruz Biotechnology, CA, USA). The membranes were further probed with HRP-conjugated secondary anti-sera (Santa Cruz Biotechnology) and visualized by ECL solution (Millipore, MA, USA) and a cooled CCD camera system (ATTO Technology, Tokyo, Japan).

| Animals
All experiments were carried out with 6-to 8-week-old male BALB/c and C57BL/6 mice weighing 19-23 g (Orient Bio, Seongnam-Si, Gyeonggi-Do, Korea). These animals were bred and maintained at 25 ± 2°C on the standard mouse diet (LabDiet, St. Louis, MO, USA). All animal experiments were approved by the Kangwon National University Institutional Animal Care and Use Committee (KIACUC-130325-1).

| Preparation of SJE
SJE was prepared by Korea Food Research Institute. Saeu-jeot (1 kg) was mixed with ethanol (99.99%) and suspended overnight at 25°C.
It was filtered through a coarse filter (No. 1, Advantec) and concentrated by vacuum evaporation. The concentrate was filtered a second time through a fine filter (No. 2, Advantec). The filtrate was adjusted to pH 7.4 with 1.0 or 0.1 mol/L NaOH. The concentration of the final SJE was adjusted to 100 mg/ml with distilled water.

| Treatment of mice with SJE
A total of 200 mg of prepared SJE/kg body weight was administered by gavage (2 ml/kg per each diluted with 8 ml/kg normal saline) to the experimental animals for 6 weeks (once a day, 5 days a week).
The animals were irradiated a day after the final feeding.

| Irradiation
Whole-body irradiation of the experimental animals was carried out in a gamma chamber (Gammacell 40 Exactor, Ottawa, Ontario,

Canada) obtained from Radiation Health Research Institute, Korea
Hydro and Nuclear Power Co., Ltd. Animals were exposed to 2-Gy gamma radiation.

| Body and spleen weight
In order to monitor any changes at the whole-body level, we weighed mice immediately following irradiation. In this study, body weight is expressed as the rate of weight gain after irradiation. Spleen weight is expressed as a proportion of body weight (spleen mass [g]/body mass [g]).

| Measurement of white and red blood cells and platelets
Following irradiation, blood was collected from the subclavian vein under ether anesthesia. Whole blood was collected using an EDTAcontaining tube (SARSTEDT, Germany); and the number of white blood cells, red blood cells, and platelets were counted using an automatic cell counter (SEAC, Italy).

| Micronuclei analysis
Micronucleus assay was used to assess chromosome damage as described previously (Kim et al., 2019). Briefly, acridine orange (Merck, Germany) was dissolved in distilled water (1 mg/ml), and 10 µl of this solution was placed on pre-heated (~70°C) clean glass microscope slides. Approximately 3 µl of peripheral blood was obtained from the subclavian vein and smeared onto a glass slide. MnPCEs was analyzed by a fluorescence microscope, and at least 1,000 polychromatic erythrocytes in the peripheral blood were scored per mouse for each data point. Five mice were included in each experimental group.

| Statistical analysis
All data are presented as the mean ± standard deviation. For comparisons of MnPCEs between groups, data were analyzed using the SAS 9.3 statistical package. All data were analyzed using the nonparametric Kruskal-Wallis test.

| Effects of SJE on body and spleen weights and blood cells
There was no significant difference in the rate of body weight gain and spleen-to-body weight between the mice after irradiation with and without SJE extract (Table 1).
Hematological test results showed that there was no significant difference in leukocytes, erythrocytes, and platelets between control group and SJE group (Table 2).

| SJE inhibits NLRP3 inflammasome activation
To assess the effects of SJE alone on NLRP3 inflammasome activation, various dosages of extract (2.5%-10%) were used to treat LPSprimed BMDMs. No mature IL-1β was detected in the supernatant, although secreted pro-IL-1β was observed with the higher concentrations of SJE. Instead, we assessed the inhibitory effects of SJE on inflammasome activation by various known activators. Surprisingly, SJE inhibited IL-1β secretion after activating BMDMs with a wellcharacterized NLRP3 inflammasome activator ATP (Figure 1a).
In addition, we elucidated the inhibitory effects of SJE on NLRP3 inflammasome activation with other NLRP3 activators, such as nigericin (NG), calcium chloride (CaCl 2 ), and aluminum potassium sulfate (Alum). SJE attenuated the production of mature IL-1β resulting Note: Data are presented as mean ± SD.

TA B L E 2
Hematological analyses of mice after irradiation with and without SJE from NG-, CaCl 2 -, and Alum-induced NLRP3 inflammasome activation, although the degree of inhibition by SJE varied (Figure 1b).  Figure 2, like SJE, sea salt inhibited IL-1β secretion from ATP-or NG-treated BMDMs. In addition, NaCl also blocked IL-1β maturation resulting from NLRP3 inflammasome activation. One of the major mineral components, magnesium, also inhibited IL-1β production. However, KCl did not alter IL-1β maturation induced by ATP or NG (Figure 2d). Thus, the sodium and magnesium in the sea salt, the major component of saeu-jeot, inhibit NLRP3 inflammasome activation.

| Frequency of micronuclei
Micronuclei are additional small nuclei formed by excluding chromosome fragments or the whole chromosome during mitosis.
Therefore, the micronucleus rate reflects chromosome breakage mitotic apparatus damage. Based on this, we compared the MnPCEs frequencies between treatment and control groups. There was a significant difference between control and SJE treatment in both mouse strains (*p < .05) (Figure 3).

| Inflammatory responses induced by irradiation cause chromosome damage
Radiation-induced inflammatory responses have been proposed to affect chromosome instability (Licandro et al., 2013;Multhoff & Radons, 2012). The inflammasome drives the cleavage of the inflammatory cytokine precursor pro-IL-1β into the active form IL-1β, which can then be secreted and induce inflammatory responses that lead to DNA damage (Multhoff & Radons, 2012;Wiseman & Halliwell,).
During this process, inflammasomes NLRP3, NLRC4, and AIM2 have key roles in the activation of inflammatory cytokine precursors (Jin & Flavell, 2010). The NLRP3 inflammasome drives DNA damage after oxidative and genotoxic stress; it sustains the ROS-mediated oxidation of DNA (Licandro et al., 2013).

| SJE inhibited NLRP3 inflammasome activation
In this study, we observed that SJE inhibited IL-1β secretion after the activation of BMDMs with a well-characterized NLRP3 inflammasome activator. We also elucidated the inhibitory effects of SJE on NLRP3 inflammasome activation by other NLRP3 activators, such as NG, CaCl 2 , and Alum. SJE attenuated mature IL-1β production resulting from NG-, CaCl 2 -, and Alum-induced NLRP3 inflammasome activation, thereby inhibiting NLRP3 inflammasome activation,

F I G U R E 1 Effects of SJE on NLRP3
inflammasome activation. BMDMs were primed by LPS in RPMI medium containing 10% FBS and antibiotics for 3 h. Cells were supplied with RPMI medium in the present with the indicated percentages of SJE with/without NLRP3 inflammasome activators, ATP, nigericin (NG), and CaCl 2 for 1 h, or aluminum potassium sulfate (Alum) for 3 h, and were analyzed for IL-1β secretion. Cell culture supernatants (Sup) and cell lysates (Lys) were analyzed by immunoblot assay as indicated although the degree of inhibition varied. Similar to SJE, sea salt inhibited IL-1β secretion from ATP-or NG-treated BMDMs. NaCl also blocked the IL-1β maturation resulting from NLRP3 inflammasome activation. Magnesium, a major mineral in sea salt, inhibited IL-1β production. However, potassium (KCl), which is an NLRP3 inflammasome inhibitor, did not alter the IL-1β maturation induced by ATP or NG. Therefore, the sodium and magnesium in sea salt, a major component of saeu-jeot, inhibit NLRP3 inflammasome activation.

| SJE treatment reduced MnPCE frequency
There is within-species genetic variation in radiation sensitivity. The range of radiation LD 50/30 values ranges from 4 to 6.5 Gy among laboratories; it is confounded by differences in the age at exposure, radiation quality, and unidentifiable environmental factors (Grahn & Hamilton, 1957;Hamasaki et al., 2007). BALB/c mice are considered more radiosensitive than C57BL/6 mice; the difference in LD 50/30 F I G U R E 2 Effects on the sea salt and its ingredients of SJE on the NLRP3 inflammasome activation. LPS-primed BMDMs were stimulated with NLRP3 inflammasome activator, ATP or nigericin (NG), in the presence of the indicated concentration of sea salt (a), NaCl (b), MgCl 2 (c), or KCl (d). After 1 h, cell culture supernatants (Sup) and cell lysates (Lys) were collected and analyzed by immunoblot assay as indicated values between the two strains is approximately 1.3 Gy (Hamasaki et al., 2007).
As expected, there were significantly more radiation-induced

| Anti-oxidative effects of the ingredients in SJE
Other factors may have contributed to the decrease in MnPCEs in the SJE treatment groups because MnPCEs mainly result from cellular oxidative stress (Iyer & Lehnert, 2000). Chitosan, a natural nontoxic biopolymer produced by the deacetylation of chitin, is a major component of crustacean shells (e.g., crabs, shrimp, and crawfish).
Chito-oligosaccharides (COS) are the enzymatic or chemical hydrolysis products of chitosan or chitin; they may be generated during chitosan fermentation. Many recent studies have reported that chitin and its derivatives, such as COS, inhibit DNA and protein oxidation.
In addition, intracellular glutathione levels and direct intracellular radical scavenging by mouse macrophages were increased in the presence of chitin and chitin derivatives, exerting an overall protective effect against cellular oxidative stress (Lodhi et al., 2014;Ngo et al., 2009;Park & Kim, 2010). During the 6-week SJE treatment, oxidative stress in hematopoietic tissue was decreased, which could reduce radiation-induced chromosome damage. Some ingredients in SJE are free radical scavengers that reduce the damage caused by ionizing radiation-induced free radicals. Further studies are necessary to determine which ingredients contribute to the radioprotective effects of saeu-jeot and to elucidate the mechanisms by which such effects are mediated.

| SJE is a radioprotective agent
To be clinically useful, a good radioprotective agent must be effective, readily available, specific, and well tolerated (Duraković, 1993).
Saeu-jeot, a fermented seafood commonly consumed in Korea, is an inexpensive, readily available agent that can be used for an extended period of time; thus, it may serve as a good radioprotective agent.
Our results support this application.

ACK N OWLED G EM ENTS
This work was supported by Kangwon National University (520160310).

CO N FLI C T O F I NTE R E S T
The authors declare that there are no potential conflicts of interest regarding the publication of this article.

DATA AVA I L A B I L I T Y S TAT E M E N T
All datasets obtained and analyzed during this study are available from the corresponding author on reasonable request.